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Five multidrug-resistant nonclonally related Enterobacteriaceae isolates were recovered in Belgium in 2010 from three patients who had been hospitalized in Pakistan, Montenegro, and Serbia/Kosovo. New Delhi metallo-␤-lactamase (NDM-1) was detected in each of the isolates in addition to several extended-spectrum ␤-lactamases (CTX-M-15, SHV-12), plasmidic cephalosporinases (CMY-16, CMY-58), rRNA methylases (ArmA, RmtB), and Qnr genes (qnrA6, qnrB1, qnrB2). One patient died from uncontrolled sepsis, while the two others recovered. No secondary cases occurred in any of the hospitals.

Four screening assays aimed for rapid detection of carbapenemase production from Gram-negative bacterial isolates, i.e., the Neo-Rapid Carb kit (Rosco Diagnostica A/S), the Rapidec Carba NP test (bioMérieux SA), the ␤ Carba test (Bio-Rad Laboratories N.V.), and a homemade electrochemical assay (BYG Carba test) were evaluated against a panel comprising 328 clinical isolates (Enterobacteriaceae [n ϭ 198] and nonfermentative Gram-negative bacilli [n ϭ 130]) with previously characterized resistance mechanisms to carbapenems. Among Enterobacteriaceae isolates, the BYG Carba test and the ␤ Carba test showed excellent sensitivities (respectively, 100% and 97.3%) and specificities (respectively, 98.9% and 97.7%). The two other assays yielded poorer performances with sensitivity and specificity of 91.9% and 83.9% for the Rapidec Carba NP test and of 89.2% and 89.7% for the Neo-Rapid Carb kit, respectively. Among Pseudomonas spp., sensitivities and specificities ranged, respectively, from 87.3% to 92.7% and from 88.2% to 94.1%. Finally, all tests performed poorly against Acinetobacter spp., with sensitivities and specificities, respectively, ranging from 27.3% to 75.8% and from 75 to 100%. Among commercially available assays, the ␤ Carba test appeared to be the most convenient for routine use and showed the best overall performances, especially against OXA-48-like producers. The excellent performance of the BYG Carba test against Enterobacteriaceae was confirmed (100% sensitivity and 98.9% specificity).

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We report the first description of the metallo-␤-lactamase VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in Enterobacter cloacae 11236, which was isolated from blood specimens of a patient with colonic adenocarcinoma in Belgium. bla VIM-31 was found on a class 1 integron located on a self-transferable but not typeable 42-kb plasmid. Compared to values published elsewhere for VIM-2, the purified VIM-31 enzyme showed weaker catalytic efficiency against all the tested betalactam agents (except for ertapenem), resulting from lower k cat (except for ertapenem) and higher K m values for VIM-31. The worldwide spread of metallo-␤-lactamases (i.e., VIM, IMP, and NDM enzymes) in carbapenem-resistant Gram-negative bacteria presently constitutes a major public health issue.So far, 33 distinct bla VIM alleles have been described (www .lahey.org/Studies) for a variety of Gram-negative opportunistic pathogens, but only VIM-1, VIM-2, VIM-4, VIM-7, and VIM-27 enzymes (7,12,15,22,31) have been characterized biochemically and structurally. While VIM-1-derived enzymes have been reported largely for Enterobacteriaceae from around the world, especially from Greece but also recently from Belgium (4), VIM-2 has been associated mostly with Pseudomonas aeruginosa (32). The occurrence of VIM-2 has nevertheless also been reported for clinical isolates of Enterobacteriaceae from Asia (17, 24, 39), Mexico (28), Argentina (16), and Tunisia (10). Moreover, a recent report showed the presence of VIM-2 in Enterobacter ludwigii isolated from sewage water of a hospital, highlighting the involvement of environmental bacteria as potential reservoirs for the dissemination of clinically relevant metallo-␤-lactamase resistance genes (37). In many subclass B1 (e.g., IMP and NDM) and B2 (e.g., CphA) metallo-␤-lactamases, the amino acid at position 224 (a conserved Lys residue), close to the active site, plays an important role in substrate binding (2, 27). In the case of VIM enzymes, Lys224 is not present and is replaced by His224 in VIM-1 and by Tyr224 in VIM-2. The side chains of histidine and tyrosine residues are much shorter than that of a lysine residue, thereby preventing the interaction of these two amino acids with the carboxylate moiety (C 4 or C 3 ) of the substrate. For VIM enzymes, it is hypothesized that Arg228, with its long side chain, may replace Lys224 in interacting with the substrate (15,22).We report here the detection and characterization of VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in an Enterobacter cloacae clinical isolate recovered from an elderly patient in Belgium. MATERIALS AND METHODSBacterial strains and antimicrobial susceptibility. Enterobacter cloacae 11236 was recovered in June 2011 from blood culture specimens from a patient hospitalized in Brussels, Belgium. The isolate was identified to the species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with a Microflex LT mass spectrometer (Bruker Daltonik GmbH, Leipzi...